Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Treatment of cancer includes administering a compound of formula I, for
example desritonavir, to a subject. In particular, treatment of breast
cancer is described.

Claims:

1. A method of treating cancer in a subject, the method comprising
administering to the subject a therapeutically effective amount of a
compound of formula I: ##STR00009## wherein each R is independently H
or C1-6 alkyl, or a pharmaceutically acceptable salt form thereof

2. The method of claim 1, wherein the compound of formula I is:
##STR00010## or a pharmaceutically acceptable salt form thereof.

Description:

[0003] This disclosure relates to the treatment of cancers, for example,
breast cancer, by methods that include administration of a compound of
formula I, for example, desthiazolyl ritonavir. In particular, methods of
treating ER.sup.+, triple negative and her2.sup.- breast cancers and lung
cancer are described.

BACKGROUND

[0004] Cancer is now the second leading cause of death in the United
States. In 1995, cancer accounted for 23.3% of all deaths in the United
States. See, e.g., U.S. Dept. of Health and Human Services, National
Center for Health Statistics, Health United States 1996-97 and Injury
Chartbook 117 (1997).

[0005] Cancer is now primarily treated with one or a combination of three
types of therapies: surgery; radiation; and chemotherapy. Surgery
involves the bulk removal of diseased tissue. While surgery is sometimes
effective in removing tumors located at certain sites, for example, in
the breast, colon, and skin, it cannot be used in the treatment of tumors
located in other areas, such as the backbone, nor in the treatment of
disseminated neoplastic conditions such as leukemia. Radiation therapy
involves the exposure of living tissue to ionizing radiation causing
death or damage to the exposed cells. Side effects from radiation therapy
may be acute and temporary, while others may be irreversible.
Chemotherapy involves the disruption of cell replication, cell
metabolism, or cell invasion, motility and metastasis. It is used most
often in the treatment of breast, lung, and testicular cancer as well as
hematologic malignancies such as leukemia and myeloma. One of the main
causes of failure in this treatment of cancer is the development of drug
resistance by the cancer cells, a serious problem that may lead to
recurrence of disease or even death.

[0006] In one example, new treatments for breast cancer are being
developed based on the understanding that the progression of breast
cancer requires resistance to cell death; however, the mechanisms by
which breast cancer cells acquire this attribute is not well understood.
One way in which breast cancer cells are thought to accomplish this
resistance to cell death is to activate Akt. Activated Akt can act
through multiple pathways to promote resistance to cell death and is
therefore considered to be a regulator of cancer cell survival. While the
molecular mechanisms by which Akt is activated are not well understood,
recent research had determined that an oral HIV protease inhibitor drug,
ritonavir, FDA approved to treat AIDS, exhibits activity against breast
cancer in a mouse model of mammary cancer and blocks Akt activation (see,
e.g., U.S. Publication No. 2007/0009593, incorporated by reference
herein). Ritonavir is one example of an emerging approach to the
treatment of cancer.

SUMMARY

[0007] Provided herein is a method of treating cancer comprising
administering to a subject an effective amount of a compound of formula
I:

##STR00001##

wherein each R is independently H or C1-6 alkyl. In one embodiment,
the compound of formula I is:

##STR00002##

this compound is also known as desthiazolyl ritonavir (des-ritonavir or
M1, as used herein). M1, for example, is used in the synthesis of
ritonavir and is also a minor metabolite of ritonavir (see, e.g.,
Denissen, J. F. et al., Drug Metabolism and Disposition 25(4):489-501
(1997).) The compound of formula I may be more active as an inhibitor of
cancer cell lines (e.g., breast cancer) than ritonavir (see Examples
1-7).

[0008] In some embodiments, the subject is a human. In certain
embodiments, the subject is post-menopausal.

[0010] In some embodiments, the subject has a cancer associated with
resistance to a known anticancer drug regime. Such drug regimes, for
example, can be selected from one or more of taxol, Herceptin, Avastin,
fluouracil, and epirubicin. In other embodiments, the cancer comprises
cells that express a P-glycoprotein (MDR), a multidrug
resistance-associated protein (MRP), or a breast cancer resistance
protein (BCRP).

[0011] Without being bound by theory, the compound of formula I may treat
cancer through inhibition of a cytochrome P450, such as an epoxygenase
(e.g., CYP3A4 or CYP3A5). In another embodiment, the compound of formula
I may reduce the amount of one or more epoxyeicosatrienoic acids in at
least one tumor cell of the subject relative to a tumor cell in a subject
not administered a compound of formula I. In some embodiments, the
epoxyeicosatrienoic acid is one or more of 5,6-epoxyeicosatrienoic acid;
8,9-epoxyeicosatrienoic acid; 11,12-epoxyeicosatrienoic acid; and
14,15-epoxyeicosatrienoic acid. In certain embodiments, the
epoxyeicosatrienoic acid is 14(R),15(S)-epoxyeicosatrienoic acid or
14(S),15(R)-epoxyeicosatrienoic acid. In some embodiments, the compound
of formula I reduces the amount of phosphorylated Akt in at least one
tumor cell of the subject relative to a tumor cell in a subject not
administered a compound of formula I. In other embodiments the compound
of formula I inhibits Hsp90.

[0013] Also provided herein is a pharmaceutical composition comprising a
compound of formula I and ritonavir, or a pharmaceutically acceptable
salt form thereof. In some embodiments, the composition further comprises
a carrier, excipient, or diluent. The ratio of the compound of formula I
to ritonavir can be about 20:1. In some embodiments, the ratio of the
compound of formula I to ritonavir can be about 2.75:1; about 2:1; or
about 1.81:1.

[0014] A method of inducing apoptosis in a cell is provided herein, the
method comprising contacting the cell with a therapeutically effective
amount of a compound of formula I, or a pharmaceutically acceptable salt
form thereof.

[0015] A method of reducing the amount of one or more of an
epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin dependent kinase, an
ER, or her2 in a cell is also provided herein, the method comprising
contacting the cell with a therapeutically effective amount of a compound
of formula I, or a pharmaceutically acceptable salt form thereof.

[0016] Further provided herein is a method of inhibiting a cytochrome P450
in a cell, the method comprising contacting the cell with a
therapeutically effective amount of a compound of formula I, or a
pharmaceutically acceptable salt form thereof.

[0017] Also provided herein is a method of reducing the amount of
phosphorylated Akt in a cell, the method comprising contacting the cell
with a therapeutically effective amount of a compound of formula I, or a
pharmaceutically acceptable salt form thereof.

[0018] A method of inhibiting Hsp90 in a cell is provided herein, the
method comprising contacting the cell with a therapeutically effective
amount of a compound of formula I, or a pharmaceutically acceptable salt
form thereof.

[0019] Further provided herein is a method of treating cancer in a
subject, the method comprising inhibiting CYP3A4 in the subject. A method
of treating cancer in a subject, is also provided wherein the method
comprises administering to the subject a pharmaceutical that inhibits
CYP3A4.

[0020] Provided herein is a kit comprising a compound of formula I, or a
pharmaceutically acceptable salt form thereof. In some embodiments, the
kit further comprises instructions for treating a subject.

[0021] The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent from
the description and drawings, and from the claims.

[0042] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as is commonly understood by one of ordinary
skill in the art to which this disclosure belongs. All patents,
applications, published applications, and other publications are
incorporated by reference in their entirety. In the event that there is a
plurality of definitions for a term herein, those in this section prevail
unless stated otherwise.

[0043] As used herein, "administration" refers to delivery of a compound
of formula I by any external route, including, without limitation, IV,
intramuscular, SC, intranasal, inhalation, transdermal, oral, rectal,
sublingual, and parenteral administration. It does not include the
administration of a prodrug, i.e., any compound (whether itself active or
inactive) that is converted chemically in vivo into a biologically active
compound of formula I following administration of the prodrug to a
patient. For example, administration, as used herein, does not include
production of a compound of formula I via in vivo metabolism of
ritonavir.

[0044] The term "contacting" means bringing at least two moieties
together, whether in an in vitro system or an in vivo system.

[0045] The expression "effective amount," when used to describe an amount
of compound applied in a method, refers to the amount of a compound that
achieves the desired pharmacological effect or other effect, for example
an amount that inhibits the abnormal growth or proliferation, or induces
apoptosis of cancer cells, resulting in a useful effect.

[0046] The terms "treating" and "treatment" mean causing a therapeutically
beneficial effect, such as ameliorating existing symptoms, preventing
additional symptoms, ameliorating or preventing the underlying metabolic
causes of symptoms, postponing or preventing the further development of a
disorder and/or reducing the severity of symptoms that will or are
expected to develop.

[0047] As used herein, "subject" (as in the subject of the treatment)
means both mammals and non-mammals. Mammals include, for example, humans;
non-human primates, e.g. apes and monkeys; cattle; horses; sheep; rats;
mice; pigs; and goats. Non-mammals include, for example, fish and birds.

[0048] As used herein, "alkyl" carbon chains, if not specified, should be
broadly interpreted, for example to encompass substituted or
unsubstituted, straight, branched, unsaturated, and cyclic "chains."

II. Methods of Use

[0049] Provided herein are methods of treating cancer in a subject by
administering to the subject a therapeutically effective amount of a
compound of formula I:

##STR00003##

wherein each R is independently H or C1-6 alkyl; or a
pharmaceutically acceptable salt form thereof. In some embodiments, the
compound of formula I is:

##STR00004##

This compound is also known as desthiazolyl ritonavir or M1. Although all
stereochemical species are contemplated in this application, in some
embodiments, the compound according to formula I has the following
structure:

[0066] Cancers may be solid tumors that may or may not be metastatic.
Cancers may also occur, as in leukemia, as a diffuse tissue. Thus, the
term "tumor cell," as provided herein, includes a cell afflicted by any
one of the above identified disorders. In some embodiments, a subject may
have cancer (e.g., breast cancer, lymphoma, HD, or leukemia) and
increased cardiac risk factors, such as those for whom anthracycline is
not an appropriate treatment.

[0067] In some embodiments, the subject is a human. In certain
embodiments, the subject is a female human. In other embodiments, the
subject is post-menopausal. In some embodiments, the subject is a male
human.

[0068] In some embodiments, the subject has a cancer associated with
resistance to a known anticancer drug regime, e.g., wherein the cancer
comprises cells that express a P-glycoprotein (MDR), a multidrug
resistance-associated protein (MRP), or a breast cancer resistance
protein (BCRP). In certain embodiments the anticancer drug regime is
selected from one or more of Taxol, Abraxane, Herceptin, Avastin,
fluorouracil, and epirubicin. Also the anticancer drug regime could
include statin drugs.

[0069] A method of treating cancer using a compound of formula I may be
combined with existing methods of treating cancers, for example by
chemotherapy, irradiation, or surgery (e.g., oophorectomy). In certain
embodiments, a compound of formula I can be used as a radiation
sensitizer in the treatment of head and neck cancer, lung cancer, and
glioma (see, e.g., Gupta, A. and Mueschel, R. Cancer Res.
65(18):8256-8265 (2005)). In some embodiments, a compound of formula I
can be administered before, during, or after another anticancer agent or
treatment.

[0070] In some embodiments (e.g., when compositions comprising a compound
of formula I are administered in conjunction with another pharmaceutical
(e.g. an anticancer agent or ritonavir), one can create a synergistic
effect among the agents administered and thereby improve the outcome for
a patient. In some embodiments, a compound of formula I (or a
pharmaceutically acceptable salt form thereof) can be administered in
combination with (i.e., before, during, or after) administration of a
pain relief agent (e.g., a nonsteroidal anti-inflammatory drug such as
celecoxib or rofecoxib), an antinausea agent, or an additional anticancer
agent (e.g., paclitaxel, docetaxel, doxorubicin, daunorubicin,
epirubicin, fluorouracil, melphalan, cis-platin, carboplatin,
cyclophosphamide, mitomycin, methotrexate, mitoxantrone, vinblastine,
vincristine, ifosfamide, teniposide, etoposide, bleomycin, leucovorin,
taxol, Herceptin, Avastin, Abraxane, cytarabine, dactinomycin, interferon
alpha, streptozocin, prednisolone, irinotecan, gemcitabine, pemetrexed,
sulindac, 5-fluorouracil, capecitabine or procarbazine). In certain
embodiments, the anticancer agent is paclitaxel or docetaxel. In other
embodiments, the anticancer agent is cisplatin or irinotecan.

[0071] Non-limiting examples of combination therapies include the
following. In the treatment of her2+ breast cancer, for example, a
compound of formula I can be administered in combination with Herceptin
or Tykerb. In the treatment of lung cancer, head and neck cancer, colon
cancer, and breast cancer, for example, a compound of formula I can be
administered in combination with cetuximab, erlotinib, or gefitinib. In
the treatment of colon cancer, for example, a compound of formula I can
be administered in combination with Avastin or Folfox combination. In the
treatment of prostate cancer, for example, a compound of formula I can be
administered in combination with docetaxel. In the treatment of breast
cancer, for example, a compound of formula I can be administered in
combination with Abraxane. In the treatment of renal cancer, for example,
a compound of formula I can be administered in combination with
sorafenib, sunitinib, or temsirolimus. In the treatment of melanoma or
glioma, for example, a compound of formula I can be administered in
combination with temozolomide. In the treatment of mantle cell lymphoma
or myeloma, for example, a compound of formula I can be administered in
combination with Velcade. In the treatment of diffuse large B cell
lymphoma, for example, a compound of formula I can be administered in
combination with rituximab-CHOP therapy (cyclophosphamide, doxorubicin,
vincristine, and prednisone). In the treatment of chronic myelogenous
leukemia, for example, a compound of formula I can be administered in
combination with imatinib or dasatinib. In the treatment of epithelial
malignancies, for example, a compound of formula I can be administered in
combination with ketoconazole or itraconazole.

[0072] In some embodiments, a compound of formula I can be combined with
an HIV protease inhibitor, such as those that function in non-cross
resistant mechanisms (e.g., nelfinavir). In another embodiment, a
compound of formula I can be combined with a drug that is not metabolized
though CYP3A, e.g., gemcitabine. In other embodiments, a compound of
formula I may be combined with a hormonal therapy, e.g., tamoxifen,
fulvestrant, gosereline, and exemestane.

[0073] In some embodiments, a compound of formula I is combined with
ritonavir. In some embodiments, the ratio of a compound of formula I to
ritonavir can be about 20:1 (e.g., about 18:1; about 16:1; about 15.5:1;
about 15:1; about 12:1; about 11.75:1; about 10:1; about 8:1; about
7.25:1; about 6:1; about 5:1; about 4.6:1; about 4:1; about 3:1; about
2.75:1; about 2.5:1; about 2:1; about 1.8:1; about 1.75:1; about 1.5:1;
about 1.25:1; and about 1:1). In some embodiments, the ratio of a
compound of formula I to ritonavir is about 2.75:1. In some embodiments,
the ratio is about 2:1. In some embodiments, the ratio is about 1.8:1. In
some embodiments, the ratio of a compound of formula I to ritonavir can
be about 1:20 (e.g., about 1:18; about 1:16; about 1:15.1; about 1:15;
about 1:12; about 1:11.75; about 1:10; about 1:8; about 1:7.25; about
1:6; about 1:5; about 1:4.6; about 1:4; about 1:3; about 1:2.75; about
1:2.5; about 1:2; about 1:1.5).

[0074] In some embodiments, a compound of formula I can be administered
with a statin drug, acetylsalicylic acid, an imid drug (e.g., revlimid),
an Hsp90 inhibitor (e.g., 17-AAG), and/or any drug currently in clinical
development for the treatment of cancer (e.g., vitamin D3).

[0076] In some embodiments, administration of a compound of formula I and
an additional therapeutic agent can produce a synergistic effect. This
effect can be demonstrated through the development of a combination index
(CI). In certain embodiments, the index can be calculated as a function
of the fraction of cells affected according to the procedure of Chou and
Talalay, Advance Enz. Regul. (1985) 22: 27-55. This is a well-known test
that evaluates coefficient interactions against a range of cell death
proportions. For example, if treatment with drug A results in 30% cell
death and treatment with drug B results in 50% cell death, than it would
be expected that the combination of the two drugs would result in 65%
cell death. Accordingly, if the ratio of the predicted cell death to that
measured upon combination of the drugs is less than one, then a
synergistic effect is observed.

[0077] A method of inhibiting a cytochrome P450 in a cell is also provided
herein, the method comprising contacting the cell with a therapeutically
effective amount of a compound of formula I. In some embodiments, the
cytochrome P450 is an epoxygenase. In certain embodiments, the
epoxygenase is selected from the CYP3A subfamily (e.g., CYP3A4 and
CYP3A5). In some embodiments, the epoxygenase is CYP3A4. In other
embodiments, the cytochrome P450 is selected from the CYP1A subfamily,
CYP1B subfamily, CYP2C subfamily (e.g., CYP2C8 and CYP2C9), CYP2J2, CYP4F
subfamily, CYP4A subfamily and CYP19A1. The method of inhibiting
cytochrome P450 in a cell may be performed by contacting the cell with a
compound according to formula I, or a pharmaceutically acceptable salt
form thereof, in vitro, thereby inducing inhibition of cytochrome P450 of
a cell in vitro. Uses of such an in vitro method of inhibiting cytochrome
P450 include, but are not limited to use in a screening assay (for
example, wherein a compound according to formula I is used as a positive
control or standard compared to compounds of unknown activity or potency
in inhibiting cytochrome P450). In some embodiments thereof, cytochrome
P450 is inhibited in a cancer cell. See Examples 10-15.

[0078] Inhibition of a cytochrome P450 can be determined by, for example,
incubating CYP3A4 or CYP3A5 Supersomes® with cytochrome b5, and 5
μM arachidonic acid (AA) with or without NADPH. The minus NADPH
reactions can be used as a control for AA oxidation. By measurement of
epoxyeicosatrienoic acid products using electron capture APCI MS/MS, it
can be determined whether CYP3A4 or CYP3A5 synthesis of
epoxyeicosatrienoic acid regio- and stereoisomers is inhibited by a
compound of formula I. In another example, inhibition of CYP2C8, CYP2D6
and CYP2J2 can be tested using model pharmacologic substrates (e.g.,
astemizole and ebastine for 2J2).

[0079] The method of inhibiting cytochrome P450 in a cell may be
performed, for example, by contacting a tumor cell with a compound
according to formula I, in vivo, thereby inhibiting a cytochrome P450 in
a subject in vivo. The contacting is achieved by causing a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof, to be present in the subject in an amount effective to achieve
inhibition of the cytochrome P450. This may be achieved, for example, by
administering an effective amount of a compound according to formula I,
or a pharmaceutically acceptable salt form thereof, to a subject. Uses of
such an in vivo method of inhibiting a cytochrome P450 include, but are
not limited to use in methods of treating a disease or condition, wherein
inhibiting of the cytochrome P450 is beneficial. In some embodiments
thereof, the cytochrome P450 is inhibited in a cancer cell, for example
in a patient suffering from cancer. The method is preferably performed by
administering an effective amount of a compound according to formula I,
or a pharmaceutically acceptable salt form thereof, to a subject who is
suffering from cancer.

[0080] Also provided herein is a method of treating cancer in a subject,
the method comprising inhibiting CYP3A4 in the subject. In some
embodiments, a method of treating cancer in a subject can include
administering to the subject a pharmaceutical that inhibits CYP3A4. As
above, the methods can be performed in vivo or in vitro and includes
contacting a cell (e.g., a cancer cell) with a pharmaceutical capable of
inhibiting CYP3A4. In some embodiments, the pharmaceutical is a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof

[0081] Further provided herein is a method of inducing cell cycle arrest
and/or apoptosis in a cell. The method includes contacting the cell with
a therapeutically effective amount of a compound of formula I, or a
pharmaceutically acceptable salt form thereof. The method of inducing
cell-cycle arrest and/or apoptosis of a cell may be performed by
contacting the cell with a compound according to formula I, or a
pharmaceutically acceptable salt form thereof, in vitro, thereby inducing
cell-cycle arrest and/or apoptosis of a cell in vitro. Uses of such an in
vitro method of inducing cell-cycle arrest and/or apoptosis include, but
are not limited to use in a screening assay (for example, wherein a
compound according to formula I is used as a positive control or standard
compared to compounds of unknown activity or potency in inducing
cell-cycle arrest and/or apoptosis). In some embodiments thereof, the
cell-cycle arrest and/or apoptosis is induced in a cancer cell.

[0082] Induction of apoptosis can be determined using the Annexin
V-FITC/PI apoptosis detection kit (Oncagene, Boston, Mass.). For example,
after plating at 5×105 in a 100-mm plate, cancer cells can be
grown for 48 hours in complete medium in presence of a drug, including a
compound or formula 1, and a control, such as vehicle (DMSO). The
culture and drug exposure conditions for the apoptosis assays can be done
across a range of 5 to 60 μmol/L drug. Cells can be harvested by
trypsin treatment, washed with complete medium to neutralize the trypsin,
and stained with PI and Annexin V-FITC. The events can then be analyzed
using FACScan analysis and CellQuest software (Becton Dickinson). See
Example 2.

[0083] The method of inducing cell-cycle arrest and/or apoptosis of a cell
may be performed, for example, by contacting a tumor cell with a compound
according to formula I, in vivo, thereby inducing cell-cycle arrest
and/or apoptosis in a subject in vivo. The contacting is achieved by
causing a compound according to formula I, or a pharmaceutically
acceptable salt form thereof, to be present in the subject in an amount
effective to achieve cell-cycle arrest and/or apoptosis. This may be
achieved, for example, by administering an effective amount of a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof, to a subject. Uses of such an in vivo method of inducing
cell-cycle arrest and/or apoptosis include, but are not limited to use in
methods of treating a disease or condition, wherein inducing cell-cycle
arrest and/or apoptosis is beneficial. In some embodiments thereof, the
cell-cycle arrest and/or apoptosis is induced in a cancer cell, for
example in a patient suffering from cancer. The method is preferably
performed by administering an effective amount of a compound according to
formula I, or a pharmaceutically acceptable salt form thereof, to a
subject who is suffering from cancer.

[0084] A method of reducing the amount of one or more of an
epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin dependent kinase, an
ER, or her2 in a cell is provided herein, the method comprising
contacting the cell with a therapeutically effective amount of a compound
of formula I. The amount of one or more of an epoxyeicosatrienoic acid,
Hsp90, a cyclin, a cyclin dependent kinase, an ER, or her2 in the treated
cell is reduced relative to a cell in a subject not administered a
compound of formula I. In some embodiments, an epoxyeicosatrienoic acid
is selected from 5,6-epoxyeicosatrienoic acid; 8,9-epoxyeicosatrienoic
acid; 11,12-epoxyeicosatrienoic acid; 14,15-epoxyeicosatrienoic acid; and
mixtures thereof. An epoxyeicosatrienoic acid may exist as either of two
stereoisomers, e.g., 14(R),15(S)-epoxyeicosatrienoic acid or
14(S),15(R)-epoxyeicosatrienoic acid. The method of reducing the amount
of one or more of an epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin
dependent kinase, an ER, or her2 in a cell may be performed by contacting
the cell with a compound according to formula I, or a pharmaceutically
acceptable salt form thereof, in vitro, thereby reducing the amount of
one or more of an epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin
dependent kinase, an ER, or her2 of a cell in vitro. Uses of such an in
vitro method of reducing the amount of one or more of an
epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin dependent kinase, an
ER, or her2 include, but are not limited to use in a screening assay (for
example, wherein a compound according to formula I is used as a positive
control or standard compared to compounds of unknown activity or potency
in reducing the amount of one or more epoxyeicosatrienoic acids). In some
embodiments thereof, the amount of one or more epoxyeicosatrienoic acids
is reduced in a cancer cell.

[0085] The method of reducing the amount of one or more of an
epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin dependent kinase, an
ER, or her2 in a cell may be performed, for example, by contacting a
cell, e.g., a tumor cell, with a compound according to formula I, in
vivo, thereby reducing the amount of one or more epoxyeicosatrienoic
acids in the cell in vivo. The contacting is achieved by causing a
compound according to formula I, or a pharmaceutically acceptable salt
form thereof, to be present in a subject in an amount effective to
achieve a reduction in the amount of one one or more of an
epoxyeicosatrienoic acid, Hsp90, a cyclin, a cyclin dependent kinase, an
ER, or her2. This may be achieved, for example, by administering an
effective amount of a compound according to formula I, or a
pharmaceutically acceptable salt form thereof, to a subject. Uses of such
an in vivo method of reducing the amount of one or more
epoxyeicosatrienoic acids include, but are not limited to use in methods
of treating a disease or condition, wherein reduction in the amount of
one or more epoxyeicosatrienoic acids is beneficial. In some embodiments
thereof, the amount of one or more of an epoxyeicosatrienoic acid, Hsp90,
a cyclin, a cyclin dependent kinase, an ER, or her2 is reduced in a
cancer cell, for example in a patient suffering from cancer. The method
is preferably performed by administering an effective amount of a
compound according to formula I, or a pharmaceutically acceptable salt
form thereof, to a subject who is suffering from cancer.

[0086] Further provided herein is a method of reducing the amount of
phosphorylated Akt (pAkt) in a cell, the method comprising contacting the
cell with a therapeutically effective amount of a compound of formula I.
The amount of pAkt in the treated cell is reduced relative to a cell in a
subject not administered a compound of formula I. The method of reducing
the amount of pAkt in a cell may be performed by contacting the cell with
a compound according to formula I, or a pharmaceutically acceptable salt
form thereof, in vitro, thereby reducing the amount of pAkt of a cell in
vitro. Uses of such an in vitro method of reducing the amount of pAkt
include, but are not limited to use in a screening assay (for example,
wherein a compound according to formula I is used as a positive control
or standard compared to compounds of unknown activity or potency in
reducing the amount of pAkt). In some embodiments thereof, the amount of
pAkt is reduced in a cancer cell.

[0087] The method of reducing the amount of pAkt in a cell may be
performed, for example, by contacting a cell, e.g., a tumor cell, with a
compound according to formula I, in vivo, thereby reducing the amount of
pAkt in a cell in vivo. The contacting is achieved by causing a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof, to be present in a subject in an amount effective to achieve a
reduction in the amount of pAkt. This may be achieved, for example, by
administering an effective amount of a compound according to formula I,
or a pharmaceutically acceptable salt form thereof, to a subject. Uses of
such an in vivo method of reducing the amount of pAkt include, but are
not limited to use in methods of treating a disease or condition, wherein
reduction in the amount of pAkt is beneficial. In some embodiments
thereof, the amount of phosphorylated Akt is reduced in a cancer cell,
for example in a patient suffering from cancer. The method is preferably
performed by administering an effective amount of a compound according to
formula I, or a pharmaceutically acceptable salt form thereof, to a
subject who is suffering from cancer.

[0088] Reduction of phosphorylated Akt can be evaluated, for example, by a
Western blot of cells treated with a compound of formula I for 24 and 48
h and evaluating for Akt/pAkt. Student's t-test can be used to assess
differences in total Akt and pAkt, comparing a compound of formula I,
vehicle treated cells (e.g., DMSO), and other anticancer agents (e.g.,
ritonavir). Any appropriate cell line may be used, for example, any of
the NCI 60 cell lines, such as MCF7, T47D, MDA-MB-231 and MDA-MB-436.

[0089] A method of inhibiting Hsp90 in a cell is also provided herein, the
method comprising contacting the cell with a therapeutically effective
amount of a compound of formula I. The method of inhibiting Hsp90 in a
cell may be performed by contacting the cell with a compound according to
formula I, or a pharmaceutically acceptable salt form thereof, in vitro,
thereby inducing inhibition of Hsp90 of a cell in vitro. Uses of such an
in vitro method of inhibiting Hsp90 include, but are not limited to use
in a screening assay (for example, wherein a compound according to
formula I is used as a positive control or standard compared to compounds
of unknown activity or potency in inhibiting Hsp90). In some embodiments
thereof, Hsp90 is inhibited in a cancer cell.

[0090] The method of inhibiting Hsp90 in a cell may be performed, for
example, by contacting a cell, e.g., a tumor cell, with a compound
according to formula I, in vivo, thereby inhibiting Hsp90 in a subject in
vivo. The contacting is achieved by causing a compound according to
formula I, or a pharmaceutically acceptable salt form thereof, to be
present in a subject in an amount effective to achieve inhibition of
Hsp90. This may be achieved, for example, by administering an effective
amount of a compound according to formula I, or a pharmaceutically
acceptable salt form thereof, to a subject. Uses of such an in vivo
method of inhibiting Hsp90 include, but are not limited to use in methods
of treating a disease or condition, wherein inhibiting of Hsp90 is
beneficial. In some embodiments thereof, Hsp90 is inhibited in a cancer
cell, for example in a patient suffering from cancer. The method is
preferably performed by administering an effective amount of a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof, to a subject who is suffering from cancer.

[0091] Inhibition of Hsp90 can be evaluated by surface plasmon resonance
studies of a compound of formula I, using a CM-5 Biacore chip on which
Hsp90 (Stressgen, Ann Arbor) has been immobilized. This method can also
be used to evaluate the chaperoning activity of a compound of formula I
by evaluation of luciferase refolding. See Examples 4 and 9.

[0092] Further provided herein is a method of increasing the amount of
reactive oxygen species (ROS) in a cell, the method comprising contacting
the cell with a therapeutically effective amount of a compound of formula
I. The amount of ROS in the treated cell is increased relative to a cell
in a subject not administered a compound of formula I. The method of
increasing the amount of ROS in a cell may be performed by contacting the
cell with a compound according to formula I, or a pharmaceutically
acceptable salt form thereof, in vitro, thereby increasing the amount of
ROS of a cell in vitro. Uses of such an in vitro method of increasing the
amount of oxygen species include, but are not limited to use in a
screening assay (for example, wherein a compound according to formula I
is used as a positive control or standard compared to compounds of
unknown activity or potency in increasing the amount of ROS). In some
embodiments thereof, the amount of ROS is increased in a cancer cell.

[0093] The method of increasing the amount of ROS in a cell may be
performed, for example, by contacting a cell, e.g., a tumor cell, with a
compound according to formula I, in vivo, thereby increasing the amount
of ROS in a subject in vivo. The contacting is achieved by causing a
compound according to formula I, or a pharmaceutically acceptable salt
form thereof, to be present in a subject in an amount effective to
achieve an increase in the amount of ROS. This may be achieved, for
example, by administering an effective amount of a compound according to
formula I, or a pharmaceutically acceptable salt form thereof, to a
subject. Uses of such an in vivo method of increasing the amount of ROS
include, but are not limited to use in methods of treating a disease or
condition, wherein an increase in the amount of ROS is beneficial. In
some embodiments thereof, the amount of ROS is increased in a cancer
cell, for example in a patient suffering from cancer. The method is
preferably performed by administering an effective amount of a compound
according to formula I, or a pharmaceutically acceptable salt form
thereof, to a subject who is suffering from cancer.

[0094] Production of ROS can be analyzed using flow cytometry. For
example, induction of ROS, including peroxide and superoxide, can be
studied in cancer cell line. Cells can be seeded in 6 well plates
(˜80,000 cells/well) using the appropriate medium, and exposed to a
drug for 24 hours. Subsequently, the medium can be removed from the wells
and the cells can then be exposed to DHE (2 μM in PBS) or
Carboxy-H2-DCFDA (10 μM in PBS) for 30 minutes. Cells can then be
trypsinized, washed two times with PBS and analyzed by flow cytometry.
Additional cells can be exposed to H2O2 (250 μM) for 2 hours
as a positive control for ROS production. See Example 3.

III. Pharmaceutical Compositions

[0095] Provided herein is a compound of formula I:

##STR00006##

wherein each R is independently H or C1-6 alkyl; or a
pharmaceutically acceptable salt form thereof. In some embodiments, the
compound of formula I is:

##STR00007##

and pharmaceutically acceptable salt forms thereof. This compound is also
known as desthiazolyl ritonavir (des-ritonavir or M1) is a minor
metabolite of ritonavir. Ritonavir is a known agent in the treatment of
HIV and its use as an anticancer agent has been studied (see, e.g., U.S.
Publication No. 2007/0009593, incorporated herein by reference). A
compound of formula I can be synthesized using conventional techniques
using readily available starting materials. In general, a compound of
formula I is conveniently obtained via standard organic chemistry
synthesis methods. For example, two such methods are described in U.S.
Pat. Nos. 5,616,720 and 6,407,252.

[0096] Although all stereochemical species are contemplated in this
application, in some embodiments, the compound according to formula I has
the following structure:

##STR00008##

[0097] The term "pharmaceutically-acceptable salt" refers to salts which
possess toxicity profiles within a range that affords utility in
pharmaceutical applications. Pharmaceutically unacceptable salts may
nonetheless possess properties such as high crystallinity, which may
render them useful, for example in processes of synthesis, purification
or formulation of compounds described herein. In general the useful
properties of the compounds described herein do not depend critically on
whether the compound is or is not in a salt form, so unless clearly
indicated otherwise (such as specifying that the compound should be in
"free base" or "free acid" form), reference in the specification to a
compound of formula I should be understood as encompassing salt forms of
the compound, whether or not this is explicitly stated.

[0100] All of these salts may be prepared by conventional means from the
corresponding compound according to formula I by reacting, for example,
the appropriate acid or base with a compound according to formula I.
Preferably the salts are in crystalline form, and preferably prepared by
crystallization of the salt from a suitable solvent. A person skilled in
the art will know how to prepare and select suitable salt forms for
example, as described in Handbook of Pharmaceutical Salts: Properties,
Selection, and Use By P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002).

[0101] Provided herein are pharmaceutical compositions comprising a
compound of formula I. The pharmaceutical compositions provided herein
contain a compound of formula I in an amount that is useful in the
treatment of cancer.

[0102] In some embodiments, the pharmaceutical composition further
comprises a pharmaceutically acceptable carrier, excipient, or diluent.
Pharmaceutical carriers suitable for administration of the compounds
provided herein include any such carriers known to those skilled in the
art to be suitable for the particular mode of administration.
Pharmaceutically acceptable carriers, excipients, and diluents include,
but are not limited to, ion exchangers, alumina, aluminum stearate,
lecithin, self-emulsifying drug delivery systems (SEDDS) such as
d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used
in pharmaceutical dosage forms such as Tweens or other similar polymeric
delivery matrices, serum proteins, such as human serum albumin, buffer
substances such as phosphates, glycine, sorbic acid, potassium sorbate,
partial glyceride mixtures of saturated vegetable fatty acids, water,
salts or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts,
colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,
cellulose-based substances, polyethylene glycol, sodium carboxymethyl
cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block
polymers, and wool fat. Cyclodextrins such as α-, β, and
γ-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-b-cyclodextrins, or other solubilized derivatives can
also be advantageously used to enhance delivery of compounds of the
formulae described herein. In some embodiments, the carrier, excipient,
or diluent is a physiologically acceptable saline solution.

[0103] The compounds according to formula I can also be administered in
combination with existing methods of treating cancers, for example by
chemotherapy, irradiation, or surgery. In some embodiments, a compound of
formula I can be administered before, during, or after another anticancer
agent or treatment. In some embodiments (e.g., when compositions
comprising a compound of formula I are administered in conjunction with
another anticancer agent or ritonavir), one can create a synergistic
effect among the agents administered and thereby improve the outcome for
a patient. In some embodiments, a compound of formula I (or a
pharmaceutically acceptable salt form thereof) can be administered in
combination with (i.e., before, during, or after) administration of a
pain relief agent (e.g., a nonsteroidal anti-inflammatory drug such as
celecoxib or rofecoxib), an antinausea agent, anti-diarrheal or an
additional anticancer agent (e.g., paclitaxel, docetaxel, doxorubicin,
daunorubicin, epirubicin, fluorouracil, melphalan, cis-platin,
carboplatin, cyclophosphamide, mitomycin, methotrexate, mitoxantrone,
vinblastine, vincristine, ifosfamide, teniposide, etoposide, bleomycin,
leucovorin, taxol, Herceptin, Avastin, cytarabine, dactinomycin,
interferon alpha, streptozocin, prednisolone, irinotecan, sulindac,
5-fluorouracil, capecitabine or procarbazine). In certain embodiments,
the anticancer agent is paclitaxel or docetaxel. In other embodiments,
the anticancer agent is cisplatin or irinotecan. In some embodiments, a
compound of formula I is administered in combination with (i.e., before,
during, or after) ritonavir.

[0104] Also provided herein is a pharmaceutical composition comprising a
compound of formula I and ritonavir, or a pharmaceutically acceptable
salt form thereof. In some embodiments, the composition further comprises
a carrier, excipient, or diluent. The ratio of the compound of formula I
to ritonavir can be about 20:1 (e.g., about 18:1; about 16:1; about
15.5:1; about 15:1; about 12:1; about 11.75:1; about 10:1; about 8:1;
about 7.25:1; about 6:1; about 5:1; about 4.6:1; about 4:1; about 3:1;
about 2.75:1; about 2.5:1; about 2:1; about 1.8:1; about 1.75:1; about
1.5:1; about 1.25:1; and about 1:1). In some embodiments, the ratio of
the compound of formula I to ritonavir can be about 2.75:1; about 2:1; or
about 1.81:1.

[0106] The concentration of a compound of formula I in a pharmaceutical
composition will depend on absorption, inactivation and excretion rates
of the compound, the physicochemical characteristics of the compound, the
dosage schedule, and amount administered as well as other factors known
to those of skill in the art. For example, the amount that is delivered
is sufficient to treat ER.sup.+ breast cancer, as described herein. In
another embodiment, the amount that is delivered is sufficient to treat
triple negative breast cancer.

[0107] The pharmaceutical composition may be administered at once, or may
be divided into a number of smaller doses to be administered at intervals
of time. It is understood that the precise dosage and duration of
treatment is a function of the disease being treated and may be
determined empirically using known testing protocols or by extrapolation
from in vivo or in vitro test data. It is to be noted that concentrations
and dosage values may also vary with the severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time according
to the individual need and the professional judgment of the person
administering or supervising the administration of the compositions, and
that the concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed compositions.

[0108] Absolute bioavailability can be determined by measuring the AUC
achieved with a particular dosage form and route of administration as
compared to that of an IV dosage form of a solubilized formulation of the
drug. A poorly bioavailable drug has an absolute bioavailability that is
less than about 30% (e.g., less than about 25%; less than about 20%; less
than about 15%; less than about 10%; and less than about 5%) of the IV
dosage form of the drug. On the other hand, a drug can have an acceptable
bioavailability if it can measure an absolute bioavailability of greater
than about 30% (e.g., greater than about 35%; greater than about 40%;
greater than about 45%; greater than about 50%; greater than about 55%;
greater than about 60%; greater than about 65%; greater than about 70%;
greater than about 75%; greater than about 80%; greater than about 85%;
greater than about 90%; and greater than about 95%) as compared to an IV
dosage form of the drug. In some embodiments, an oral formulation of a
compound according to formula I can have an absolute bioavailability of
greater than about 30%.

[0109] The pharmaceutical compositions are provided for administration to
humans and animals in unit dosage forms, such as tablets, capsules,
pills, powders, granules, sterile parenteral solutions or suspensions,
and oral solutions or suspensions, and oil-water emulsions containing
suitable quantities of the compounds or pharmaceutically acceptable
derivatives thereof. The pharmaceutically therapeutically active
compounds and derivatives thereof are, in one embodiment, formulated and
administered in unit-dosage forms or multiple-dosage forms. Unit-dose
forms as used herein refers to physically discrete units suitable for
human and animal subjects and packaged individually as is known in the
art. Each unit-dose contains a predetermined quantity of the
therapeutically active compound sufficient to produce the desired
therapeutic effect, in association with the required pharmaceutical
carrier, vehicle or diluent. Examples of unit-dose forms include ampoules
and syringes and individually packaged tablets or capsules. Unit-dose
forms may be administered in fractions or multiples thereof. A
multiple-dose form is a plurality of identical unit-dosage forms packaged
in a single container to be administered in segregated unit-dose form.
Examples of multiple-dose forms include vials, bottles of tablets or
capsules or bottles of pints or gallons. Hence, multiple dose form is a
multiple of unit-doses which are not segregated in packaging.

[0110] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, or otherwise mixing an
active compound as defined above and optional pharmaceutical adjuvants in
a carrier, such as, for example, water, saline, aqueous dextrose,
glycerol, glycols, ethanol, and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be administered
may also contain minor amounts of nontoxic auxiliary substances such as
wetting agents, emulsifying agents, solubilizing agents, pH buffering
agents and the like, for example, acetate, sodium citrate, cyclodextrine
derivatives, sorbitan monolaurate, triethanolamine sodium acetate,
triethanolamine oleate, and other such agents.

[0111] Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th
Edition, 1975.

[0112] Dosage forms or compositions containing a compound of formula I in
the range of 0.005% to 100% with the balance made up from non-toxic
carrier may be prepared. Methods for preparation of these compositions
are known to those skilled in the art. The contemplated compositions may
contain 0.001%-100% active ingredient, in one embodiment 0.1-95%, in
another embodiment 75-85%.

IV. Kits

[0113] Also provided herein are kits. Typically, a kit includes a compound
of formula I. In certain embodiments, a kit can include one or more
delivery systems, e.g., for a compound of formula I, and directions for
use of the kit (e.g., instructions for treating a subject). In certain
embodiments, a kit can include a compound of formula I and one or more
additional anticancer agents. In another embodiment, a kit can include a
compound of formula I and one or more antinausea agents. In some
embodiments, the kit can include a compound of formula I and one or more
pain relief agents. In some embodiments, the kit can include a compound
of formula I and ritonavir. In some embodiments, the kit can include a
compound of formula I and a label that indicates that the contents are to
be administered to a subject resistant to an anticancer agent, such as
Herceptin. In another embodiment, the kit can include a compound of
formula I and a label that indicates that the contents are to be
administered to a subject with cells expressing a P-glycoprotein (MDR), a
multidrug resistance-associated protein (MRP), or a breast cancer
resistance protein (BCRP). In another embodiment, the kit can include a
compound of formula I and a label that indicates that the contents are to
be administered to a subject with ER.sup.- breast cancer. In another
embodiment, the kit can include a compound of formula I and a label that
indicates that the contents are to be administered to a subject with
her2.sup.- breast cancer. In a further embodiment, a kit can include a
compound of formula I and a label that indicates that the contents are to
be administered with an anticancer agent. In another embodiment, a kit
can include a compound of formula I and a label that indicates that the
contents are to be administered with an antinausea agent. In some
embodiments, a kit can include a compound of formula I and a label that
indicates that the contents are to be administered with a pain relief
agent. In a further embodiment, a kit can include a compound of formula I
and a label that indicates that the contents are to be administered with
ritonavir.

EXAMPLES

Example 1

M1 Inhibits Breast Cancer Cell Proliferation

[0114] M1 inhibition of proliferation of MCF7, T47D and MDA-MB-231 cell
lines was studied. Cells (n=2000/well) were seeded in 96 well plates with
drug or vehicle (DMSO) on D1 and after 48 h the cell number was
determined by MTT assay. Each assay was performed with 8 measurements for
each data point (See FIG. 1, wherein graph A corresponds to MCF7, graph B
corresponds to T47D, and graph C corresponds to MDA-MB-231).

[0115] The MCF7-her2 line, over-expressing the her2 oncoprotein, exhibited
an identical IC50 of 25 μM for ritonavir and M1, suggesting that
her2 can overcome the added efficacy of M1 and may promote a mechanism of
resistance to the compound of formula I. The IC50 of M1 for the
non-transformed human breast epithelial line MCF10A is 50 μM,
indicating greater selective toxicity of M1 for transformed cell line
relative to nontransformed cell lines compared to ritonavir, which
exhibits an IC50 for MCF10A of 35 μM. Remarkably, M1 does not
inhibit HIV replication in an MT-4 cell based assay (see, e.g., Denissen,
J. F. et al., Drug Metabolism and Disposition: The Biological Fate of
Chemicals 25: 489-501 (1997)). Preliminary flow cytometry apoptosis
studies of the MCF7 line treated with M1 (45 μM) exhibit two-fold
higher concentration of early apoptotic cells with M1 (28%) compared to
iso-dosed ritonavir (14%), suggesting that M1 is a more efficient inducer
of apoptosis. In addition, the IC90 values for the ER.sup.+ lines
are more than 30 μM less than ritonavir, suggesting that M1 is capable
of a higher fractional cell kill.

Example 2

Apoptosis Induced by Administration of MI

[0116] Induction of apoptosis was studied in a MCF7 breast cancer cell
line. Apoptotic cells were detected using an Annexin V-FITC/PI apoptosis
detection kit (Oncagene, Boston, Mass.). After plating at
5×105 in a 100-mm plate, the cells were grown for 48 hours in
complete medium in presence of M1, ritonavir, or vehicle (DMSO). The
culture and drug exposure conditions for the apoptosis assays were done
across a range of 5 to 60 μmol/L M1 and ritonavir. Cells were
harvested by trypsin treatment, washed with complete medium to neutralize
the trypsin, and stained with PI and Annexin V-FITC. A total of
1×104 events were analyzed per assay by FACScan analysis using
CellQuest software (Becton Dickinson). Replicate assays were done to
confirm the results.

[0117]FIG. 2 compares the induction of apoptosis of ritonavir (R) and M1.
Early apoptotic cells are exhibited in the lower right quadrant and were
compared with treatment at the ritonavir IC50 (30 μM) resulting
in a significantly higher fraction of apoptotic cells for M1 versus
ritonavir (32 vs. 17%).

[0118] As FIG. 2 and the tables below illustrate, M1 is a more potent
inducer of apoptosis than ritonavir.

[0119] A similar study was performed using T47D (ER+), MDA-MB-231 (triple
negative) and SKBR3 (ER-/Her2+) cell lines were performed as above. The
fraction of apoptotic cells was significantly higher with M1 vs.
ritonavir in the T47D (20 vs. 13%), MDA-MB-231 (40 vs. 20%), and SKBR3
lines (78 vs. 11%).

Example 3

Induction of Reactive Oxygen Species by M1

[0120] Induction of Reactive Oxygen Species (ROS), including peroxide and
superoxide, were studied in the T47D cell line. Cells were seeded in 6
well plates (˜80,000 cells/well) using the appropriate medium,
(DMEM with 10% FBS, 1% L-Glu, 1% Sodium Piruvate, and 1% Pen-strep) and
exposed to ritonavir (30 μM) and M1 (30 μM) for 24 hours.
Subsequently, the medium was removed from the wells and the cells were
exposed to DHE (2 μM in PBS) or Carboxy-H2-DCFDA (10 μM in PBS) for
30 minutes. Cells were then trypsinized, washed two times with PBS and
analyzed by flow cytometry. Additional cells were exposed to
H2O2 (250 μM) for 2 hours as a positive control for ROS
production.

[0121] As shown in FIG. 3, M1 induces ROS production in the T47D cell
line, while ritonavir does not. A major 70 kDa Hsp90 fragment is observed
with M1 treatment, but is 3-fold less abundant with ritonavir (data not
shown). This fragment has been previously associated with
menadione-induced oxidative stress (see Beck R, Verrax J, Gonze T, et al.
Hsp90 cleavage by an oxidative stress leads to its client proteins
degradation and cancer cell death. Biochemical pharmacology 2008).

Example 4

Depletion of Hsp90 and Other Proteins by M1

[0122] Breast cancer cell lines MDA-MB-231, T47D and SKBR3 were used to
study depletion of Hsp90 by M1 and ritonavir. Cells were incubated for 48
hours with 1) ritonavir (R) at its IC50 (20 μM for T47D and 40
μM for MDA-MB-231); 2) M1 at ritonavir's IC50; or DMSO (control).
Cell extracts were made according to the method of Srirangam et al., Clin
Cancer Res. 2006 Mar. 15; 12(6):1883-96. Briefly, nearly confluent cell
monolayers were treated with drug or vehicle (DMSO) for 24 or 48 h. Cells
were washed with PBS on ice and scraped in a RIPA buffer containing
protease inhibitors including MG-132 and phosphatase inhibitors. The cell
lysate protein content was determined by μBCA and equal amounts of
protein were loaded per lane. Gel electrophoresis by SDS PAGE was
performed with equal protein loading per lane (usually 30 μg) and the
separated proteins were transferred to 0.45 μm nitrocellulose filters.
The transfer was verified by Ponceau stain of the nitrocellulose filters.
The filters were probed by chemiluminescent blotting and then exposed to
X-ray film. The signal from the protein of interest was normalized to the
GAPDH signal. Student's t-test was used to determine the significance of
differences.

[0123] M1 was more potent than ritonavir in reducing Hsp90 levels in the
T47D and MDA-MB-231 lines (FIG. 4; Table 1), while neither drug reduced
Hsp90 in the SKBR3 line. M1 was also more potent in reducing total Akt,
phospho-Akt, GSK3beta, c-src, and survivin for all three breast cancer
lines (P<0.05 by Student's t test). Both M1 and ritonavir reduced the
G1/S regulatory protein Cyclin D1 in MDA-MB-231 and T47D cell lines
(P<0.05 by Student's t test), but only M1 reduced the G1/S regulator
protein in the SKBR3 cell line. For the ER+ T47D line, M1 was more potent
in reducing ER alpha (P<0.05 by Student's t test). Importantly, M1,
but not ritonavir, reduced the expression of surface HER2 in the SKBR3
line, up to 80% (P<0.05 by Student's t test).

Example 5

Inhibition of a Murine Breast Cancer Xenograft

[0124] Six-8 weeks old female nude mice (nu.sup.-/nu.sup.-) (Charles River
Laboratories International, Inc., MA) were injected into a right mammary
fat pad with 1×106 log-phase MDA-MB-231. Mice were monitored
bi-weekly for weight and tumor growth. When tumors reached a volume of
approximately 30-40 mm3 (calculated according to the formula: volume
(mm3)=(length)(width)2/2), mice were randomized into three
groups (each group n=10). One group of mice was injected daily i.p. with
ritonavir (20 mg/Kg in 100 μl of ethanol-Tween 80), the second with M1
(20 mg/Kg in 100 μl of ethanol-Tween 80), and the third with vehicle
only (100 μl of ethanol-Tween 80). The dose of drugs used in this
experiment (20 mg/Kg) is the maximum tolerated dose exhibited by nude
mice for M1. Mice were weighted and their tumors were measured twice a
week for the entire duration of the treatment (42 days).

[0125] One hour after the last drug/vehicle injection plasma, tumors,
lungs, liver, tongue, and skin were collected from each mouse. Plasma was
stored at -80° C.; half of the tissues were treated to prepare
paraffin blocks, and half were frozen in liquid nitrogen and stored at
-80° C.

[0126] As shown in FIG. 5A, M1 exhibited statistically significant (by the
Student t test) delayed tumor growth compared to mice treated with only
the vehicle. Also, when the results obtained with M1 are compared to that
from ritonavir (see FIG. 5B), M1 again exhibited a statistically
significant delay in tumor growth. Although ritonavir inhibits an
MDA-MB-231 xenograft at its MTD (40 mg/Kg), it is ineffective at half its
MTD (20 mg/Kg). In contrast, M1 tested at its MTD (20 mg/Kg) reduced the
average tumor size 2-fold at 42 days of follow-up (P<0.05 by Student's
t-test) (FIG. 5A,B). Animal weight loss did not occur with either drug
(FIG. 5C).

Example 6

M1 Inhibits Breast Cancer Cell Proliferation

[0127] M1 inhibition of proliferation of MCF7 (ER+), T47D (ER+),
MDA-MB-231 (triple negative), SKBR3 (ER-/Her2+), and the non-transformed
mammary line MCF10A were studied. Cells (n=2000/well) were seeded in 96
well plates with drug or vehicle (DMSO) on D1 and after 48 h the cell
number was determined by MTT assay. In all the breast cancer lines
tested, M1 exhibited a lower IC50 compared to ritonavir: 10, 15, 22,
and 12 μM observed with M1 vs. 23, 28, 40, and 33 μM observed with
ritonavir for the T47D, MCF7, MDA-MB-231, and SKBR3 lines, respectively
(FIG. 6; SKBR3 not shown). In contrast, M1 exhibited a higher IC50
for the non-transformed breast epithelial line MCF10A: 45 vs. 31 μM
for ritonavir (FIG. 6).

Example 7

M1 as an Anti-HER2 Agent

[0128] SKBR3 cells were plated at a density of 80,000 cells/well in 6-well
plates. After 18 hours, cells were exposed to ritonavir (33 μM),
Desthiazolyl Ritonavir (M1) (33 μM), or DMSO. After 24 hours, cells
were trypsinized, spun down, and resuspended in 50 μL of Staining
Buffer (PBS 1% BSA). Cells were then incubated with anti-HER2 Ab FITC
(Chemicon International; MAB4083F) for 30 minutes (on-ice and in the
dark). Control cells were incubated with mouse IgG1 FITC (Biolytex).
Cells were washed two times with Washing Buffer (PBS 0.1% Tween-20),
resuspended in 100 μL of PBS, and acquired using FACSCalibur flow
cytometer.

[0130] Cells were plated in 96 well plates, after overnight incubation,
ritonavir and M-1 in specific ratios or DMSO vehicle were added to the
cells and incubated for 48 hours or longer. Then MTT (thiazole blue
tetrazolium bromide) was added (0.5 mg/ml) and incubated for 2 hours
followed by centrifugation in an Allegra plate centrifuge at 2000 rpm for
5 minutes. Cell pellets were dissolved in DMSO. Absorbance at 540 nm was
determined in a colorimetric plate reader. The isobologram method of Chou
and Talalay was utilized in which the ratio of M-1 and ritonavir was
fixed according to the ratio of the IC50's and then tested across a
several log range above and below the IC50 of M-1. See Adv Enzyme
Regul. 1984; 22:27-55. The Calcusyn program calculates the Chou/Talalay
combination index.

[0131] A CI value less than 1 is indicative of a synergistic effect (Chou
& Talalay's algorithm). Results indicated that the M1/ritonavir
combination has a synergistic effect at the ratios tested (see FIGS. 8-10
and Tables 5-7).

[0132] Purified mammalian Hsp90 was attached to a CM-5 BIAcore chip
through an NHS ester linkage. After attachment of the Hsp90 and blocking
the chip with BSA, M1 drug or a 17-AAG control was circulated through a
microfluidic circuit and change in refractive index was measured by
quantification of the RU value for each drug concentration. In the case
of the 17-AAG control the binding was saturable and based on the kon
and koff measurements, the KD was estimated to be in the 1 microM
range. In contrast, although M-1 binds, the binding is not saturable, as
indicated by the rising RU value with increasing M-1 concentration. This
means that there are significant differences in the binding of M-1 and
may indicate alteration of the Hsp90 structure.

[0133]FIG. 11 indicates the presence of an association between M1 and
Hsp90.

Example 10

Effect of siRNA on Proliferation of Breast Cancer Lines

[0134] A pool of four siRNA (see Table 8) was used to inhibit the CYP3A4/5
expression in the MCF-7, T47D and MDA-MD-231 breast cancer cell lines
using transient transformation. The three breast cancer lines were
transiently transfected with non-target (Control), CYP3A4 and CYP3A5
siRNA. Proliferation of the lines was monitored using MTT assay (see FIG.
12A). The P value is less than 0.05 as compared to the non-target
control. FIG. 12B shows the semiquantitative RT-PCR indicated comparative
inhibition by siRNA in the MCF7 line. The RT-PCR analysis shows that a
90% reduction is observed in the case of CYP3A4 and a 60% reduction is
observed in the case of CYP3A5. The Samples were normalized with GAPDH
and done in triplicates.

Effect of CYP3A inhibitor Azamulin on the Proliferation of Breast cancer
Lines

[0135] The effect of Azamulin was tested in the three breast cancer lines
MCF7, T47D and MDA-MB-231. A concentration of 5 μM Azamulin was used
in this study. This concentration corresponds to the IC50 of CYP3A
inhibition for this drug. The cells were plated in a phenol red free
charcoal stripped serum compensated media. Cell proliferation was
determined after 72 hrs. See FIG. 13.

[0136] The effect of CYP3A4 inhibition on migration was tested using the
MDA-MB-231 breast cancer cell line in a Boyden chamber assay. The cells
were transfected with a non-target (NT) and CYP3A4 siRNA and the
migration efficiency on the 4th day was determined. Cells were
plated on the upper chamber (1.5×105) and porous filter (8
μM) was used to separate the two chambers. Cells were incubated for 4
hours, migrated cells were fixed, stained with eosin and methylene blue,
and counted. Both the NT and the siRNA is a pool of four different
sequences. See FIG. 14.

Example 13

CYP3A4 shRNA MCF7 Clones Show Reduced Migration

[0137] The migration efficiency of the MCF7 shRNA clones were tested in a
Boyden chamber assay and compared to shRNA containing non-target
sequences (NT). Cells were plated in the upper chamber
(1.5×105) and incubated for 6 hours using a 5% serum as a
chemoattractant. The cells were treated as described in Example 12 and
counted. The CYP3A4 shRNA sequences were designed based on two separate
target sequences. See FIG. 15.

Example 14

CYP3A4 Inhibits Anchorage Independent Growth

[0138] CYP shRNA (4-14 and 3-18) and NT clones (NT-2 and NT-6) were plated
(10,000 cells/6 well) with soft agar containing 10% serum media on a hard
agar base. The cells were incubated for 7-10 days and counted in
reference to a 10×10 grid. Five representative areas were counted
from each set, and each set was performed in triplicates. The CYP shRNA
clones exhibited increased colonies as compared to NT clones. See Table 9
and FIG. 16.

[0139] A standard scratch test assay was used to determine the effect of
CYP3A4 on the motility of breast cancer cells. Cells were plated in
6-well plates and grown to confluence. The cells were then scratched
using a pipette tip, and the scratched area was measured using Leica
application software. After 24 hours the area was measured again. The
assay is performed in triplicate (three wells per condition) and an
average of 5 pictures per well were taken per time point. CYP shRNA (4-14
and 3-18) and NT clones (NT-2 and NT-6) were studied. See FIG. 17.

[0140] Examples 10-15 above illustrate the effect of CYP3A4 regulation and
inhibition on the proliferation, adhesion, and motility of various breast
cancer cell lines.

[0141] H522 adenocarcinoma non-small cell lung cancer cell proliferation
was measured in 96-well plates by a 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) assay that measures reduction of MTT in
96-well plates (see Ohno M, Abe T. J Immunol Methods, 1991 Dec. 15;
145(1-2):199-203). The cells were exposed to 10 to 80 μmol/L
ritonavir, M1 or DMSO vehicle for 48 hours and proliferation was
subsequently quantified by MTT assay. The MTT was added to the medium at
the endpoint and the plate was incubated for 2 hours. The plate was then
centrifuged in an Allegra plate centrifuge at 2000 rpm for 5 minutes. The
medium was removed, and the pellet was dissolved in 100 μL of DMSO per
well. The pellet was dissolved using a gently vibrating platform. The
plates were read with a BioTek Synergy plate reader in colorimetric mode
at 540 nm. Each assay was performed in octuplicate for each data point.

[0142] As shown in FIG. 18, measured cell proliferation was lower in cells
exposed to M1 compared to cells exposed to ritonavir or DMSO.

Example 17

Reduction of ER.sup.+ by M1

[0143] An ER.sup.+ line, T47D, was treated with M1 or ritonavir for 24 or
48 hours. The cells were grown to confluence and treated with drug or
DMSO vehicle. The cells were scraped on ice into RIPA buffer containing
protease inhibitors including MG-132 and phosphatase inhibitors as
published (see Srirangam et al. Clincal Ca Research 2006). The RIPA
lysate was subjected to electrophoresis, blotted and probed with the
appropriate antibodies. Densitometry was performed and quantified as
shown in Table 10 and FIG. 19.

[0144] A number of embodiments of the invention have been described.
Nevertheless, it will be understood that various modifications may be
made without departing from the spirit and scope of the invention.
Accordingly, other embodiments are within the scope of the following
claims.